Influence of MTHFR polymorphism, alone or in combination with smoking and alcohol consumption, on cancer susceptibility

Abstract 5,10-methylenetetrahydrofolate reductase (MTHFR) mutations play a significant role in various types of cancers, serving as crucial regulators of folate levels in this process. Several studies have examined the effects of smoking and drinking on MTHFR-related cancers, yielding inconsistent results. Therefore, the objective of this study was to evaluate the magnitude of the effects of gene-smoking or gene-drinking interactions on cancer development. We conducted a comprehensive literature search in PubMed, Web of Science, CNKI, and Wan Fang databases up until May 10th, 2022, to identify relevant articles that met our inclusion criteria. The extracted data from these studies were used to calculate the overall odds ratio (OR) and corresponding 95% confidence interval (95% CI) using either a fixed-effect or random-effect model in Stata version 11.2. Stratified analyses were performed based on ethnicity, control group origin, and cancer classification to assess the risk of cancers associated with gene-smoking or gene-drinking interactions. Sensitivity analyses were conducted to investigate potential sources of heterogeneity, and publication bias was assessed using the Begg’s test and Egger’s test. Additionally, regression analysis was employed to explore the influence of relevant variables on heterogeneity. To evaluate the statistical correlations, analytical methods such as the false-positive report probability and the Bayesian false discovery probability were applied to assess the reliability of the findings. In our meta-analysis, a total of 47 articles were included, comprising 13,701 cases and 21,995 controls for the C677T polymorphism and 5,149 cases and 8,450 controls for the A1298C polymorphism. The results indicated a significant association between C677T polymorphism and cancer risks when combined with smoking (CT + TT vs CC, OR [95% CI] = 1.225 [1.009–1.487], p = 0.041). Stratified analysis further revealed a significant increase in liver cancer risk for individuals with the C677T when combined with smoking (liver cancer: CT + TT vs CC, OR [95% CI] = 1.564 [1.014–2.413], p = 0.043), particularly among Asian smokers (CT + TT vs CC, OR [95% CI] = 1.292 [1.007–1.658], p = 0.044). Regarding the A1298C polymorphism, an elevated risk of cancer was observed in mixed populations alone (CC + AC vs AA, OR [95% CI] = 1.609 [1.087–2.381], p = 0.018), as well as when combined with smoking (CC + AC vs AA, OR [95% CI] = 1.531 [1.127–2.080], p = 0.006). In non-drinkers, C677T polymorphism was found to be associated with esophageal cancer risk (C677T: CT + TT vs CC, OR [95% CI] = 1.544 [1.011–2.359], p = 0.044) and colon cancer risk (CC + AC vs AA, OR [95% CI] = 1.877 [1.166–3.054], p = 0.010), but there was no clear link between this polymorphism and cancer risk among drinkers. The association between the C677T polymorphism and cancer risk among smokers was found to be significant, suggesting that the combination of tobacco and the C677T polymorphism may enhance the carcinogenic process, particularly in liver cancer. However, no similar relationship was observed for the A1298C polymorphism. Interestingly, significantly increased cancer risk was observed in individuals with C677T genetic variants who were nondrinkers, but not among drinkers. These findings highlight the potential role of the C677T polymorphism in modifying cancer risk in specific contexts, such as smoking and alcohol consumption.

Abstract: 5,10-methylenetetrahydrofolate reductase (MTHFR) mutations play a significant role in various types of cancers, serving as crucial regulators of folate levels in this process.Several studies have examined the effects of smoking and drinking on MTHFR-related cancers, yielding inconsistent results.Therefore, the objective of this study was to evaluate the magnitude of the effects of gene-smoking or genedrinking interactions on cancer development.We conducted a comprehensive literature search in PubMed, Web of Science, CNKI, and Wan Fang databases up until May 10th, 2022, to identify relevant articles that met our inclusion criteria.The extracted data from these studies were used to calculate the overall odds ratio (OR) and corresponding 95% confidence interval (95% CI) using either a fixed-effect or random-effect model in Stata version 11.2.Stratified analyses were performed based on ethnicity, control group origin, and cancer classification to assess the risk of cancers associated with gene-smoking or gene-drinking interactions.Sensitivity analyses were conducted to investigate potential sources of heterogeneity, and publication bias was assessed using the Begg's test and Egger's test.Additionally, regression analysis was employed to explore the influence of relevant variables on heterogeneity.To evaluate the statistical correlations, analytical methods such as the false-positive report probability and the Bayesian false discovery probability were applied to assess the reliability of the findings.In our meta-analysis, a total of 47 articles were included, comprising 13,701 cases and 21,995 controls for the C677T polymorphism and 5,149 cases and 8,450 controls for the A1298C polymorphism.The results indicated a significant association between C677T polymorphism and cancer risks when combined with smoking (CT + TT vs CC, OR [95% CI] = 1.225 [1.009-1.487],p = 0.041).Stratified analysis further revealed a significant increase in liver cancer risk for individuals with the C677T when combined with smoking (liver cancer: CT + TT vs CC, OR [95% CI] = 1.564 [1.014-2.413],p = 0.043), particularly among Asian smokers (CT + TT vs CC, OR [95% CI] = 1.292 [1.007-1.658],p = 0.044).Regarding the A1298C polymorphism, an elevated risk of cancer was observed in mixed populations alone (CC + AC vs AA, OR [95% CI] = 1.609 [1.087-2.381],p = 0.018), as well as when combined with smoking (CC + AC vs AA, OR [95% CI] = 1.531 [1.127-2.080],p = 0.006).In non-drinkers, C677T polymorphism was found to be associated with esophageal cancer risk (C677T: CT + TT vs CC, OR [95% CI] = 1.544 [1.011-2.359],p = 0.044) and colon cancer risk (CC + AC vs AA, OR [95% CI] = 1.877 [1.166-3.054],p = 0.010), but there was no clear link between this polymorphism and cancer risk among drinkers.The association between the C677T polymorphism and cancer risk among smokers was found to be significant, suggesting that the combination of tobacco and the C677T polymorphism may enhance the carcinogenic process, particularly in liver cancer.However, no similar relationship was

Introduction
Cancer is a serious human disease that has led to high mortality rates worldwide.Nowadays, increasing evidence suggests that cancer development is the result of the combined effect of environmental and genetic factors.Single nucleotide polymorphism (SNP) is the most common and stable genetic variants, and germline sequence variation could be discovered in more than 1% of the general population, which might account for 80% of the whole genome heterogeneity [1].Based on the fundamental structure changes, researchers use SNPs expediently for genotyping to determine whether they are directly or indirectly linked to specific traits or diseases [2].
Folic acid (vitamin B9) is a crucial micronutrient that cannot be synthesized by the body and is found in dark green leafy vegetables and legumes [3].Through the action of dihydrofolate (DHF) reductase, folic acid is converted to DHF, which is then further reduced to tetrahydrofolate (THF).THF is transformed into 5,10-methylenetetrahydrofolate (5,.Under the influence of the MTHF reductase (MTHFR) enzyme, 5,10-MTHF is converted to 5-MTHF, which acts as a methyl donor in the synthesis of pyrimidines and purines.Additionally, in the methylation pathway, 5-MTHF plays a critical role by converting homocysteine (Hcy) into S-adenosylmethionine (SAM).SAM is instrumental in DNA methylation processes [4].
MTHFR is one of the most extensively studied genes, playing a crucial role in folate metabolism [5].MTHFR is located at the short arm terminus of chromosome 1 (1p36.3).The DNA sequence of this MTHFR is approximately 2.2 kb (kilobases) long and consists of 11 exons [6].The C677T (rs1801133) and A1298C (rs1801131) are the most extensively studied mutations in the MTHFR variants [7].They are located in exons 4 and 7, respectively.Both variations are associated with a reduction in enzyme activity [8,9].C677T variation is present in approximately 20-40% of the population.Research findings indicate that individuals with heterozygous (CT) and homozygous mutant (TT) genotypes exhibit enzyme activities reduced to 65% and 30%, respectively, in comparison to the wild-type individuals (CC), under the C → T condition [10].This variant involves the conversion of cytosine to thymine, leading to the substitution of methionine with valine at amino acid position 222 in the MTHFR enzyme structure [11].Additionally, this mutation introduces an HinfI restriction site.As a result, the enzyme's temperature stability is compromised, causing its efficiency to decrease by approximately half and resulting in elevated levels of Hcy in individuals with low folate intake [12].The A1298C polymorphism is located 2.1 kb downstream of C677T and results in an A to C conversion at codon 429, leading to the substitution of glutamine with alanine in the MTHFR protein.This ultimately results in decreased enzyme activity.However, compared to the C677T homozygous genotype (TT) that significantly increases plasma Hcy levels, the 1298CC genotype does not show a significant increase in Hcy levels [13].
Tobacco smoke contains a substantial quantity of carcinogens and toxic substances that have the potential to promote the development of cancer and other diseases [14].These harmful components can contribute to conditions such as asthma and trigger inflammatory responses within the body.Additionally, tobacco smoke has the ability to induce genetic mutations, playing a significant role in the underlying mechanisms of tobacco-related illnesses [15].Scientific studies have demonstrated that smoking can result in double-stranded DNA breaks, which, if left unrepaired, significantly increase the individual's risk of cancer.Moreover, smoking also influences the levels of Hcy in the body, leading to alterations in global DNA methylation [16].
Alcohol (ethanol) is also considered a carcinogen, and acetaldehyde, a metabolite of ethanol, can induce inflammation in the body, leading to the generation of reactive oxygen species and subsequent downstream effects.Moreover, acetaldehyde exhibits high reactivity with DNA and possesses various carcinogenic and genotoxic properties [17].Due to its high reactivity with DNA, acetaldehyde can potentially form DNA adducts, altering its physical properties and interfering with DNA synthesis and repair, which is one of the major contributing factors in its carcinogenic mechanism.
With a further understanding of MTHFR polymorphisms, numerous studies have been carried out to explore the relationship between MTHFR polymorphisms and cancers.However, the specific interactions between these polymorphisms and smoking or drinking have not been thoroughly evaluated.Therefore, we conducted a comprehensive metaanalysis using the largest available sample size to investigate the impact of MTHFR SNPs on cancers in conjunction with smoking or drinking.To the best of our knowledge, no previous study has systematically examined the association between MTHFR variants, smoking or drinking, and various types of cancers.We anticipate that this meta-analysis will provide valuable insights into the field of cancer prevention.
The association between C677T or A1298C polymorphism and cancer risks among smokers and drinkers was evaluated using crude ORs and 95% CIs.Various models were employed to calculate the overall OR in MTHFR polymorphisms, specifically homozygote comparison (TT vs CC; CC vs AA), heterozygote comparison (CT vs CC; AC vs AA), dominant model (TT + CT vs CC; CC + AC vs AA), and recessive model (CC vs CT + TT; AA vs AC + CC).Subgroup analyses were conducted to examine the effects of these polymorphisms based on ethnicity, source of control, and cancer types.
To assess heterogeneity across studies, two statistical indices, namely I 2 and chi-square p-value, were utilized, taking into account the variation in sample sizes.If I 2 > 50% and chi-square p-value ≤0.05, indicating significant heterogeneity among studies, a random-effects model was used to estimate the combined OR and 95% CI.Otherwise, a fixedeffect model was employed.
Meta-regression analyses were conducted to investigate potential sources of heterogeneity that could influence the results, taking into account the year of publication, ethnicity, and control group origin.Sensitivity analyses were carried out to assess the robustness of the findings by systematically removing individual studies and examining their impact on the overall results.Publication bias was assessed using the Begg funnel plot, which plots the standard error against the log (OR), and the Egger regression asymmetry test.All statistical analyses were performed using Stata software (version 11.2).A p-value below 0.05 was considered statistically significant, and all pvalues were two-sided.
To assess the credibility of significant associations, we employed the false-positive report probability (FPRP) and Bayesian false discovery probability (BFDP) as calculation methods.We set a pre-established threshold of 0.2 for FPRP and considered values below this threshold as noteworthy.Additionally, we used prior probabilities of 0.25, 0.1, 0.01, 0.001, and 0.0001 to determine the association between an odds ratio (OR) of 1.5 and cancer risk.Furthermore, we set a threshold of 0.8 for BFDP, and considered values below this threshold as noteworthy.Prior probabilities of 0.01, 0.001, and 0.00001 were used in the BFDP calculations.Similarly, we set 0.8 as the thresholds of BFDP and took 0.01, 0.001, 0.00001 as the prior probability of BFDP.It is important to note that FPRP values below 0.2 and BFDP values below 0.8 were considered significant and deserving of attention [18,19].

Identification and characteristic of studies
As depicted in Figure 1, our search strategy yielded a total of 243 studies.Through an initial screening of titles and abstracts, we excluded 82 studies that were deemed irrelevant, as well as 39 reviews, including meta-analyses, based on the exclusion criteria.Subsequently, upon careful examination of the full text of each article, we excluded 5 studies due to data overlap or duplication, 2 studies without complete full text access, and 68 studies that lacked sufficient data for calculation.Ultimately, our analysis included a total of 47 studies, comprising 44 studies with 13,701 cases and 21,995 controls for C677T polymorphism , 19 studies with 5,149 cases and 8,450 controls for A1298C polymorphism [20,22,23,25,27,31,33,36,39,41,[43][44][45][46]48,[58][59][60] (Tables 1 and 2).
In the final analysis, 36 articles investigated the relationship between C677T polymorphism and cancers among smokers, while 27 articles focused on the relationship between C677T polymorphism and cancers among drinkers.As for the A1298C gene, 19 articles were relevant, with 16 of them exploring the association between A1298C polymorphism and cancers among smokers and 12 articles examining the association between A1298C polymorphism and cancers among drinkers.

C677T polymorphism among smokers and drinkers
Among the 36 smoking-related literatures, there were 5 studies focused on lung cancer, 6 studies for gastric cancer, 6 studies for esophageal cancer, 3 studies for colorectal cancer, 2 studies for pancreatic cancer, 2 studies for liver cancer, and 2 studies for bladder cancer.The remaining types of cancers, such as breast cancer and thyroid cancer, did not have enough studies for subgroup analysis and were represented by less than one article each.In terms of control group source, 17 studies were hospital-based, and 19 studies were population-based.Regarding ethnicity, 28 studies included Asian population, 4 studies included Caucasian population, and 4 studies included mixed population.
Among the 27 alcohol-related literature, there were 4 studies focused on lung cancer, 3 studies on pancreatic cancer, 3 studies on colorectal cancer, 4 studies on gastric cancer, 2 studies on liver cancer, 2 studies on esophageal cancer, 2 studies on colon cancer, and 2 studies on rectal cancer.In terms of control group source, 16 studies were hospital-based and 11 studies were population-based.Regarding ethnicity, 20 studies included Asian population, 2 studies included Caucasian population, and 5 studies included mixed population.Table 1 provides a summary of the characteristics of the included studies for the C677T polymorphism among smokers and drinkers.

A1298C polymorphism among smokers and drinkers
Among the 15 smoking-related studies, there were 3 studies focused on lung cancer, 3 studies on colorectal cancer, and  2 studies on bladder cancer.For other types of cancer such as colon cancer, head and neck cancer, and liver cancer, there were fewer than 2 studies conducted for each, thus subgroup analysis was not performed.In terms of control group source, 7 studies were hospital-based and 8 studies were population-based.Regarding ethnicity, 8 studies included Asian population, 4 studies included Caucasian population, and 3 studies included mixed population.
For the 13 alcohol-related studies, there were 3 studies on colon cancer, 2 studies on colorectal cancer, 2 studies on rectal cancer, and others.Concerning the control group source, 7 studies were hospital-based and 6 studies were population-based.Regarding ethnicity, 10 studies included Asian population and 2 studies included mixed population.Table 2 provides the characteristics of the included studies for the A1298C polymorphism among smokers and drinkers.

Relationship between C677T polymorphism and cancer among smokers, non-smokers, drinkers, and non-drinkers
The analysis of the included articles revealed that the C677T polymorphism significantly increased overall cancer susceptibility among smokers in the dominant model (I 2 = 83.20%,CT + TT vs CC, OR [95% CI] = 1.225 [1.009-1.487],p = 0.041), However, no significant association was observed among non-smokers.In subgroup analyses, Asian smokers were to have that elevated risk of cancer with 1.292-fold in the dominant model, whereas Asian non-smokers were not.No association was observed among Caucasian smokers or non-smokers.Liver cancer risk was elevated among smokers by 1.564-fold, compared to non-smokers.Furthermore, smokers had a 1.564-fold increased risk of liver cancer compared to non-smokers.The risk of esophageal cancer was significantly increased in both smokers and non-smokers.However, no significant difference was found between smokers and non-smokers in terms of the association between the C677T polymorphism and other types of cancer such as lung cancer and gastric cancer.The detailed results are presented in Table 3.
The analysis revealed that the overall risk of cancer was higher among non-drinkers in the dominant model (I 2 = 80.50%, CT + TT vs CC, OR [95% CI] = 1.248 [1.001-1.557],p = 0.049), compared with drinkers.Subgroup analyses found that there was no significant difference between drinkers and non-drinkers in Asians, as well as a mixed population.Additionally, non-drinkers had a 1.544-fold increased risk of esophageal cancer and a 1.877-fold increased risk of colon cancer compared to drinkers.The detailed results are presented in Table 4.

Relationship between A1298C polymorphism and cancer among smokers, non-smokers, drinkers, and non-drinkers
The analysis of included articles revealed that A1298C polymorphism was irrelevant to overall cancer risk among smokers and non-smokers.However, subgroup analyses revealed a significant increase in cancer risk among the mixed population in the dominant model, with a 1.531-fold increased risk for nonsmokers and a 1.609-fold increased risk for smokers.No significant association between A1298C polymorphism and cancer risk was observed among Asian and Caucasian populations, regardless of smoking status.Detailed results can be found in Table 5.Similar to the findings for C677T polymorphism, the A1298C variation was associated with an increased cancer risk among non-drinkers in the dominant model.The analysis showed a significant association with a 1.171-fold increased risk in non-drinkers.However, no significant association was observed between A1298C polymorphism and cancer risk among drinkers.Subgroup analyses indicated that the presence of the dominant variant did not correlate with cancer risk among both smokers and nonsmokers.Please refer to Table 6 for detailed results.

Publication bias
Possible publication bias was examined based on the result of the calculation of Begg's test and Egger's test.For C677T polymorphism, some results showed publication bias based on Egger's test values (for smokers: CT + TT vs CC, p = 0.009; for non-smokers: TT vs CT + CC, p = 0.043; for drinkers: TT vs CC, p = 0.002).For A1298C polymorphism (for non-smokers: CC + AC vs AA, p = 0.043).

Sensitivity analysis
Only the homozygote comparison of smokers in the sensitivity analysis detected transformation of results for A1298C, indicating the reliability of most of our findings.To explore the sources of heterogeneity, we conducted a meta-regression using appropriate models based on I 2 and chi-square Combined action of MTHFR polymorphisms and environmental factors on cancer  9 p-values.The meta-regression analysis identified the year of publication and the region of the control group as potential sources of heterogeneity.

FPRP and BFDP tests
The FPRP and BFDP values for the C677T and A1298C polymorphisms are presented in Tables 3-6.The FPRP was utilized to assess the likelihood of significant findings in the results.Based on an OR of 1.5, with a prior probability of 0.25 and 0.1, several values were found to be less than 0.2 in almost all models.However, there were only a few BFDP estimations below 0.8 when considering a prior probability of 0.01, 0.001, and 0.000001 with an OR of 1.5.It is worth noting that only the estimations with FPRP less than 0.2 and BFDP less than 0.8 are deemed significant.These significant estimations suggest that our results should be interpreted cautiously.

Discussion
The two SNPs of most interest in MTHFR, C677T and A1298C, are both thought to be associated with possible carcinogenesis by altering the stability of the MHTFR enzyme [61].
Although the correlation between MTHFR SNPs and cancer susceptibility has been widely studied, most metaanalyses supported an association between the MHTFR SNPs and an increased risk of cancer [62][63][64].Here, as possible cancer risk factors, tobacco and alcohol consumption might have direct or indirect synergistic actions on cancers with enzymes regulated by these SNPs.We decided to use this meta-analysis to assess the association of MTHFR C677T or A1298C polymorphism alone and in combination with smoking or drinking on cancers.
Chemicals found in tobacco, such as nitrosamines, can cause DNA recombination, a process that can lead to mutations in other cancer related genes, thereby increasing the risk of the disease [65].
Our research suggests that smoking increases the overall risk of cancer in individuals with the C677T variant.However, no such effect was found in carriers of the A1298C variant.This may be related to the loss of enzyme activity caused by MTHFR mutation, where the enzyme activity of A1298C mutant is reduced to a lesser extent than C677T.Individuals carrying the TT genotype of the C677T polymorphism exhibit elevated levels of total homocysteine (tHcy) and reduced levels of folate in their serum compared to those with the CT and CC genotypes [66].These mutations lead to a reduction in the universal methyl donor SAM during folic acid metabolism, which may result in genomic instability and DNA fragmentation.Under normal conditions, cells maintain a standard methylation pattern by balancing the DNA methylation and demethylation processes.This balance is disrupted under pathological conditions such as inflammation, oxidative stress, and cancer, leading to different phenotypes [67].
Moreover, previous studies have demonstrated that hydrocarbons in tobacco reduce the bioactivity of vitamin B12 and folic acid.Smokers in the National Center for Health Statistics study also had lower levels of red blood cell folate compared to non-smokers [68].Similarly, a study showed that pregnant women exposed to smoking had lower levels of folic acid compared to women not exposed to tobacco [69].
Furthermore, a significantly elevated risk of carriers harboring the C677T variant was observed in Asian smokers while not in Caucasian smokers, which is consistent with a previous study [62].This could be attributed to the differences in the frequency of the T allele.Xie et al. in 2015 reported that there were prominent differences in T allele frequencies among Asian (0.396), Indian (0.132), Caucasian (0.326), Middle Eastern (0.201), and African (0.196) populations [63].
Therefore, it is possible that the combined effects of tobacco and C677T on cancers disrupt DNA methylation pathways through folic acid metabolism.However, some researchers have found that the cancer-promoting effect of MTHFR polymorphism may not be influenced by smoking [70,71].
Our study revealed varying combined effects of the C677T polymorphism and smoking on different types of cancer.Interestingly, the role of smoking habits in promoting esophageal cancer risk was similar among both smokers and non-smokers, suggesting that smoking may not be as significant of a factor in esophageal cancer as previously believed.However, it remains an independent factor, as the presence of the C677T polymorphism increased the risk of esophageal cancer in our study, as well as in the meta-analysis conducted by Langevin et al. [72].Consistent with previous evidence, we also observed that smoking was not a risk factor for colorectal [73] and bladder cancers [74].The combined effect of tobacco and C677T mutation on liver cancer was the strongest, 1.564 for smokers and 1.072 for nonsmoker.This finding aligns with the meta-analysis conducted by Qi et al. [75], which identified the C677T mutation as a risk factor for liver cancer.In contrast, the situation is different for esophageal cancer, as individuals with the C677T polymorphism who smoke show a significant increase in the risk of liver cancer.This observation can possibly be attributed to the fact that folate metabolism primarily takes place in the liver [76].An animal study demonstrated that a folatedeficient diet had an impact on gene expression in the liver of offspring mice but not in the colon.Additionally, smoking has been shown to exacerbate folate acid levels.This could be a significant contributing factor to the heightened risk of liver cancer associated with the combination of smoking and the C677T polymorphism.As for A1298C, the majority of studies indicate that it is not associated with an increased risk of cancer and may even have a protective effect against liver cancer [77], gastric cancer [78], and lung cancer [9], which supports the findings of our study, even when considering the influence of smoking.In brief, smoking might be an essential factor in the carcinogenesis in individuals with the C677T mutation, particularly increasing the risk of cancers in Asians, including liver cancer.On the other hand, the combined effect of smoking and A1298C polymorphism on cancers was not significant.
Alcohol is known to act as a folic acid antagonist, and excessive consumption can lead to folate deficiency due to reduced intake of food rich in micronutrients, impaired in intestinal absorption, and changes in metabolic pathways [79].Thus, we anticipated that alcohol consumption could potentially induce DNA hypomethylation through the aforementioned mechanisms, leading to cancer.When investigating the effect of alcohol consumption on cancer risk, specifically for C677T polymorphism, our findings indicated a decreased risk of cancer among drinkers compared to non-drinkers, particularly for esophageal and colorectal cancer.This finding was contrary to our initial expectations.In a previous study, Taioli et al. showed that the protective effect of C677T variant on colorectal cancer was limited to individuals who regularly consumed alcohol and might be influenced by the folate level [73].A Japanese case-control study also found a 69% lower risk of esophageal cancer in patients with the TT genotype in the heavy drinking subgroup [53].Therefore, in addition to alcohol consumption, we suspect that the reduced risk of cancer in drinkers with MHTFR variants may be due to ignoring the interaction between folic acid intake and individual MTHFR variants.Adequate folate intake could offset the lower enzymatic activity of MTHFR that may increase the MTHFR and promote DNA synthesis, while the adequate provision of methyl donors could still be ensured [80].Plenty of studies have also shown that the protective effect of the TT genotype is limited to individuals with high folic acid intake and low alcohol intake [81][82][83].However, because of lack of detailed raw data on the association between smoking, drinking, and folic acid intake, we cannot further explore the relationship between MHTFR polymorphisms and smoking-folic acid associations or drinking-folic acid associations with cancers.In mixed populations, we observed a lower risk of cancer among drinkers with the C677T variant.For the A1298C polymorphism, we only observed a decreased overall cancer risk in the dominant model.In brief, alcohol consumption may act as a protective factor for cancer incidence in individuals with MTHFR variants.The protective effect appears to be organ-specific and race-specific for the C677T polymorphism, particularly under conditions of adequate folate levels.
When interpreting the results of our meta-analysis, it is important to acknowledge several limitations.First, we focused on studying the individual associations of the C677T and A1298C polymorphisms with cancer risk, without considering their combined effects or the influence of smoking and drinking.However, the available data on the combined effects and interactions between these polymorphisms, as well as smoking and drinking, were insufficient for further analysis.Second, due to limitations in the available data, we were unable to categorize smoking and alcohol consumption into detailed subgroups based on intensity (light/medium/heavy). Instead, we defined individuals as current/ever smokers or non-smokers, as well as drinkers or non-drinkers.This simplified classification may not fully capture the potential nuances of smoking and alcohol consumption patterns.
In conclusion, our study highlights the significant influence of smoking and drinking on the relationship between MTHFR polymorphisms and cancer development.The interaction between smoking and the C677T mutation was associated with an increased risk of cancers, particularly liver cancer, and this effect was most prominent in Asian populations.However, the A1298C polymorphism did not show a significant association with cancer risk, even in the presence of tobacco exposure.Conversely, a negative association was observed between alcohol consumption and cancer risk among individuals with either the C677T or A1298C mutation.Future studies with larger sample sizes are needed to further explore the combined effects of tobacco or alcohol and MTHFR polymorphisms at varying folate levels in cancer development (Figure 2).

Figure 1 :
Figure 1: The entire flow diagram of filtering the available articles in this Study.

Table 2 :
Characteristics of the eligible studies for A1298C polymorphism among smokers, non-smokers, drinkers, and non-drinkers HB: Hospital-based; PB: Population-based; NA: not available.a dominant model (CC + AC/AA); b recessive model (AA/CC + AC).

Table 3 :
Integral analysis of the association between C677T polymorphism and cancer risk among smoking population

Table 3 :
ContinuedCombined action of MTHFR polymorphisms and environmental factors on cancer  11

Table 3 :
ContinuedCombined action of MTHFR polymorphisms and environmental factors on cancer  13

Table 4 :
Integral analysis of the association between C677T polymorphism and cancer risk among drinking populationCombined action of MTHFR polymorphisms and environmental factors on cancer  15

Table 4 :
ContinuedCombined action of MTHFR polymorphisms and environmental factors on cancer  17

Table 4 :
ContinuedBold value indicates statistically significant OR values, values with FPRP less than 0.2, and values with BFDP less than 0.8.Combined action of MTHFR polymorphisms and environmental factors on cancer  19

Table 5 :
Integral analysis of the association between A1298C polymorphism and cancer risk among smoking population

Table 5 :
ContinuedCombined action of MTHFR polymorphisms and environmental factors on cancer  21

Table 6 :
Integral analysis of the association between A1298C polymorphism and cancer risk among drinking populationCombined action of MTHFR polymorphisms and environmental factors on cancer  23

Table 6 :
ContinuedBold value indicates statistically significant OR values, values with FPRP less than 0.2, and values with BFDP less than 0.8.Combined action of MTHFR polymorphisms and environmental factors on cancer  25